1. Field of the Invention
The present invention relates generally to the field of protein engineering. More particularly, it concerns improved methods and compositions for the screening of combinatorial antibody libraries expressed in bacteria.
2. Description of Related Art
Currently recombinant therapeutic antibodies have sales of well over $10 bn/yr and with a forecast of annual growth rate of 20.9% they are projected to increase to $25 bn/yr by 2010. Monoclonal antibodies (mAbs) now comprise the majority of recombinant proteins currently in the clinic, with more than 150 products in studies sponsored by companies located worldwide (Pavlou and Belsey, 2005). In terms of therapeutic focus, the mAb market is heavily focused on oncology and arthritis, immune and inflammatory disorders, and products within these therapeutic areas are set to continue to be the key growth drivers over the forecast period. As a group, genetically engineered mAbs generally have higher probability of FDA approval success than small-molecule drugs. At least 50 biotechnology companies and all the major pharmaceutical companies have active antibody discovery programs in place.
The original method for isolation and production of mAbs was first reported at 1975 by Milstein and kohler (Kohler and Milstein, 1975), and it involved the fusion of mouse lymphocyte and myeloma cells, yielding mouse hybridomas. Therapeutic murine mAbs entered clinical study in the early 1980s; however, problems with lack of efficacy and rapid clearance due to patients' production of human anti-mouse antibodies (HAMA) became apparent. These issues, as well as the time and cost consuming related to the technology became driving forces for the evolution of mAb production technology. Polymerase Chain Reaction (PCR) facilitated the cloning of monoclonal antibodies genes directly from lymphocytes of immunized animals and the expression of combinatorial library of fragments antibodies in bacteria (Orlandi et al., 1989). Later libraries were created entirely by in vitro cloning techniques using naïve genes with rearranged complementarity determining region 3 (CDR3) (Griffiths and Duncan, 1998; Hoogenboom et al., 1998). As a result, the isolation of antibody fragments with the desired specificity was no longer dependent on the immunogenicity of the corresponding antigen. Moreover, the range of antigen specificities in synthetic combinatorial libraries was greater than that found in a panel of hybridomas generated from an immunized mouse. These advantages have facilitated the development of antibody fragments to a number of unique antigens including small molecular compounds (haptens) (Hoogenboom and Winter, 1992), molecular complexes (Chames et al., 2000), unstable compounds (Kjaer et al., 1998) and cell surface proteins (Desai et al., 1998).
During the past decade several display methods and other library screening techniques have been developed for isolating antigen specific binders from large ensembles of recombinant antibody fragments. These technologies are now widely exploited to engineer antibody fragments with high affinity and specificity. Many of these screening platforms share four key steps with the procedure for antibody generation in the in vivo immune system: first, the generation of genotypic diversity; second, the coupling of genotype to phenotype; third, the application of selective pressure; and fourth, amplification.
Phage display is currently the most widespread method for the display and selection of large collections of antibody fragments (Hoogenboom, 2002). In this methodology, a gene of interest is fused in-frame to phage genes encoding surface-exposed proteins, most commonly pIII. The gene fusions are translated into chimeric proteins in which the two domains fold independently. Phage displaying a protein with binding affinity for a ligand can be readily enriched by selective adsorption onto immobilized ligand, a process known as “panning”. The bound phage is desorbed from the surface, usually by acid elution, and amplified through infection of E. coli cells. Usually, 4-6 rounds of panning and amplification are sufficient to select for phage displaying specific polypeptides, even from a very large libraries with diversities up to 1010. The most successful application of phage display include the following: first, the de novo isolation of high-affinity human antibody fragments from nonimmune and synthetic libraries (Griffiths et al., 1994; Vaughan et al., 1996; de Haard et al., 1999; Knappik et al., 2000; Hoet et al., 2005), including antibody fragments against self antigens; second, the generation of picomolar affinity antibodies by in vitro affinity maturation (Yang et al., 1995; Schier et al., 1996; Lu et al., 2003) and third, the discovery of antibody fragments with unique properties from non-immune (Chames et al., 2000; Huie et al., 2001) and immune libraries from animal or human donors (Moulard et al., 2002; Kramer et al., 2005).
Ribosome and mRNA display represent another method for the display and screening of libraries of antibody fragments. The concept relays on the stable formation of a complex of antibody fragment and its encoding mRNA (Lipovsek and Pluckthun, 2004). In ribosome display, the link between antibody fragment and encoding mRNA is made by the ribosome, which at the end of translating this mRNA is made to stop without releasing the polypeptide. The ternary complex as a whole is used for the selection. In mRNA display, there is a covalent bond between the antibody fragment protein and the mRNA which is established via puromycin as an adaptor molecule. These display methods are carried out entirely in vitro.
In microbial cell display screening is carried out by flow cytometry. In particular, Anchored Periplasmic Expression (APEx) is based on anchoring the antibody fragment on the periplasmic face of the inner membrane of E. coli followed by disruption of the outer membrane, incubation with fluorescently labeled antigen and sorting of the spheroplasts. APEx was used for the affinity maturation of antibody fragments (Harvey et al., 2004; Harvey et al., 2006). In one study over 200-fold affinity improvement was obtained after only two rounds of screening.
Nonetheless, all high throughput antibody screening technologies available to-date rely on microbial expression of antibody fragments. The use of antibody fragments rather than intact or full length IgGs, in the construction and screening of libraries has been dictated by limitations related to the expression of the much larger IgGs in microorganisms. IgG libraries have never before been expressed or screened using microorganisms such as bacteria or yeasts. As a result the isolation of antigen binding proteins has been carried out exclusively using antibody fragments that are smaller and much easier to produce. Once isolated such antibody fragments have to then fused to vectors that express full length immunoglobulins which in turn are expressed preferentially in mammalian cells such as CHO cells.
Antibody fragments, including Fabs and especially single chain Fv's (scFv), pose several limitations: (1) Antibody fragments often exhibit low affinity for the target antigen. Unlike IgG or IgM antibodies antibody fragments are monovalent and therefore they cannot capitalize on avidity effects for stronger binding to antigens (Pini and Bracci, 2000). Thus, the isolation of recombinant antibody fragments to targets that cannot be recognized with high affinity e.g. carbohydrates, is problematic. (2) Antibody fragments generally exhibit lower thermodynamic stability than their corresponding full length IgG counterparts (Worn and Pluckthun, 2001). (3) Because of their small size and their lack of an Fc region, antibody fragments exhibit very short circulation half-lives compared to full length IgG proteins (Milenic et al., 1991). Therefore for the vast majority of clinical applications antibody fragments isolated from combinatorial libraries have to be converted to full length IgG molecules.
The need for isolating intact IgG molecules from combinatorial libraries has long been recognized. In an attempt to establish a platform for the isolation of recombinant IgGs, researchers recently displayed small libraries of IgGs on the surface of mammalian cells. After homologous integration of a single-gene copy in each cell, the population was sorted by flow cytometry to obtain single selected clones (W. D. Shen, Amgen, cited by Hoogenboom (2005)). Nevertheless, this technology is time-consuming, cumbersome, and expensive and is not amenable to the screening of large libraries comprising of many tens of millions of different antibody proteins. The present invention overcomes these limitations by avoiding the need for expression of IgG libraries in mammalian cells. Instead, the inventors have devised methodologies for the screening of libraries produced and secreted by E. coli bacteria.
E. coli possesses a reducing cytoplasm that is unsuitable for the folding of proteins with disulfide bonds which accumulate in an unfolded or incorrectly folded state (Baneyx and Mujacic, 2004). In contrast to the cytoplasm, the periplasm of E. coli is maintained in an oxidized state that allows the formation of protein disulfide bonds. Notably, periplasmic expression has been employed successfully for the expression of antibody fragments such as Fvs, scFvs, Fabs or F(ab′)2s (Kipriyanov and Little, 1999). These fragments can be made relatively quickly in large quantities with the retention of antigen binding activity. However, because antibody fragments lack the Fc domain, they do not bind the FcRn receptor and are cleared quickly; thus, they are only occasionally suitable as therapeutic proteins (Knight et al., 1995). Until recently, full-length antibodies could only be expressed in E. coli as insoluble aggregates and then refolded in vitro (Boss et al., 1984; Cabilly et al., 1984). Clearly this approach is not amenable to the high throughput screening of antibody libraries since, with the current technology it is not possible to refold millions or tens of millions of antibodies individually. The expression of full length IgG antibodies in secreted form in E. coli was reported only recently (Simmons et al., 2002). However, there is no information on whether antibody libraries consisting of many different antibodies can also be expressed in bacteria. Equally importantly, the prior art does not disclose any methods for isolating E. coli cells expressing a particular IgG with specificity towards a desired antigen from a vast excess of cells expressing IgGs of unrelated specificities.
E. coli expressed antibodies are not glycosylated, and fail to bind to complement factor 1q (C1q) or FcγRI and thus cannot elicit complement activation or mediate the recruitment of macrophages. However, aglycosylated Fc domains can bind to the neonatal Fc receptor efficiently (FcRn). Consequently bacterially expressed aglycosylated antibodies exhibit serum persistence and pharmacokinetics similar to those of fully glycosylated IgGs produced in human cells.